Javeed, N., Tardi, N. J., Maher, M., Singari, S. and Edwards, K. A. (2015). Controlled expression of Drosophila homeobox loci using the Hostile takeover system. Dev Dyn 244: 808-825. PubMed ID: 26017699.Summary:
Hostile takeover (Hto) is a Drosophila protein trapping system that allows the investigator to both induce a gene and tag its product. The Hto transposon carries a GAL4-regulated promoter expressing an exon encoding a FLAG-mCherry tag. Upon expression, the Hto exon can splice to a downstream genomic exon, generating a fusion transcript and tagged protein. Using rough-eye phenotypic screens, Hto inserts were recovered at eight homeobox or Pax loci: cut, Drgx/CG34340, Pox neuro, araucan, shaven/D-Pax2, Zn finger homeodomain 2, Sex combs reduced (Scr), and the abdominal-A region. The collection yields diverse misexpression phenotypes. Ectopic Drgx was found to alter the cytoskeleton and cell adhesion in ovary follicle cells. Hto expression of cut, araucan, or shaven gives phenotypes similar to those of the corresponding UAS-cDNA constructs. The cut and Pox neuro phenotypes are suppressed by the corresponding RNAi constructs. The Scr and abdominal-A inserts do not make fusion proteins, but may act by chromatin- or RNA-based mechanisms. It is concluded that Hto can effectively express tagged homeodomain proteins from their endogenous loci; the Minos vector allows inserts to be obtained even in transposon cold-spots. Hto screens may recover homeobox genes at high rates because they are particularly sensitive to misexpression.

Deshpande, S.A., Yamada, R., Mak, C.M., Hunter, B., Soto Obando, A., Hoxha, S. and Ja, W.W. (2015).Acidic food pH increases palatability and consumption and extends Drosophila lifespan. J Nutr [Epub ahead of print]. PubMed ID: 26491123Summary: Despite the prevalent use of Drosophila as a model in studies of nutrition, the effects of fundamental food properties, such as pH, on animal health and behavior are not well known. This study examined the effect of food pH on adult Drosophilalifespan, feeding behavior, and microbiota composition and tested the hypothesis that pH-mediated changes in palatability and total consumption are required for modulating longevity. The effect of buffered food (pH 5, 7, or 9) was measured on male gustatory responses (proboscis extension), total food intake, and male and female lifespan. The effect of food pH on germfree male lifespan was also assessed. Changes in fly-associated microbial composition as a result of food pH were determined by 16S ribosomal RNA gene sequencing. Male gustatory responses, total consumption, and male and female longevity were additionally measured in the taste-defective Pox neuro (Poxn) mutant and its transgenic rescue control. An acidic diet increases Drosophila gustatory responses (40-230%) and food intake (5-50%) and extends survival (10-160% longer median lifespan) compared with flies on either neutral or alkaline pH food. Alkaline food pH shifts the composition of fly-associated bacteria and results in greater lifespan extension (260% longer median survival) after microbes are eliminated compared with flies on an acidic (50%) or neutral (130%) diet. However, germfree flies live longer on an acidic diet (5-20% longer median lifespan) compared with those on either neutral or alkaline pH food. Gustatory responses, total consumption, and longevity are unaffected by food pH in Poxn mutant flies. Food pH can directly influence palatability and feeding behavior and affect parameters such as microbial growth to ultimately affect Drosophila lifespan. Fundamental food properties altered by dietary or drug interventions may therefore contribute to changes in animal physiology, metabolism, and survival.

Minocha, S., Boll, W. and Noll, M. (2017). Crucial roles of Pox neuro in the developing ellipsoid body and antennal lobes of the Drosophila brain. PLoS One 12(4): e0176002. PubMed ID: 28441464Summary:
The paired box gene Pox neuro (Poxn) is expressed in two bilaterally symmetric neuronal clusters of the developing adult Drosophila brain, a protocerebral dorsal cluster (DC) and a deutocerebral ventral cluster (VC). This study shows that all cells that express Poxn in the developing brain are postmitotic neurons. During embryogenesis, the DC and VC consist of only 20 and 12 neurons that express Poxn, designated embryonic Poxn-neurons. The number of Poxn-neurons increases only during the third larval instar, when the DC and VC increase dramatically to about 242 and 109 Poxn-neurons, respectively, virtually all of which survive to the adult stage, while no new Poxn-neurons are added during metamorphosis. Although the vast majority of Poxn-neurons express Poxn only during third instar, about half of them are born by the end of embryogenesis, as demonstrated by the absence of BrdU incorporation during larval stages. At late third instar, embryonic Poxn-neurons, which begin to express Poxn during embryogenesis, can be easily distinguished from embryonic-born and larval-born Poxn-neurons, which begin to express Poxn only during third instar, (1) by the absence of Pros, (ii) their overt differentiation of axons and neurites, and (iii) the strikingly larger diameter of their cell bodies still apparent in the adult brain. The embryonic Poxn-neurons are primary neurons that lay out the pioneering tracts for the secondary Poxn-neurons, which differentiate projections and axons that follow those of the primary neurons during metamorphosis. The DC and the VC participate only in two neuropils of the adult brain. The DC forms most, if not all, of the neurons that connect the bulb (lateral triangle) with the ellipsoid body, a prominent neuropil of the central complex, while the VC forms most of the ventral projection neurons of the antennal lobe, which connect it ipsilaterally to the lateral horn, bypassing the mushroom bodies. In addition, Poxn-neurons of the VC are ventral local interneurons of the antennal lobe. In the absence of Poxn protein in the developing brain, embryonic Poxn-neurons stall their projections and cannot find their proper target neuropils, the bulb and ellipsoid body in the case of the DC, or the antennal lobe and lateral horn in the case of the VC, whereby the absence of the ellipsoid body neuropil is particularly striking. Poxn is thus crucial for pathfinding both in the DC and VC. Additional implications of these results are discussed.

Murata, S., Brockmann, A. and Tanimura, T. (2017). Pharyngeal stimulation with sugar triggers local searching behavior in Drosophila. J Exp Biol [Epub ahead of print]. PubMed ID: 28684466Summary:Foraging behavior is essential for all organisms to find food containing nutritional chemicals. A hungry fly of Drosophila melanogaster performs local searching behavior after drinking a small amount of sugar solution. Using video tracking this study examined how the searching behavior is regulated in D. melanogaster. A small amount of highly concentrated sugar solution was found to induce a long-lasting searching behavior. After the intake of sugar solution, a fly moved around in circles and repeatedly returned to the position where the sugar droplet had been placed. The non-nutritious sugar, D-arabinose, but not the non-sweet nutritious sugar, D-sorbitol, was effective in inducing the behavior, indicating that sweet sensation is essential. Furthermore, pox-neuro mutant flies with no external taste bristles showed local searching behavior, suggesting the involvement of the pharyngeal taste organ. Experimental activation of pharyngeal sugar-sensitive gustatory receptor neurons by capsaicin using the Gal4/UAS system induced local searching behavior. In contrast, inhibition of pharyngeal sugar-responsive gustatory receptor neurons abolished the searching behavior. Together these results indicate that in Drosophila the pharyngeal taste-receptor neurons trigger searching behavior immediately after ingestion.

Pox neuro is expressed in mother cells that give rise to p-es organs, and embryos deleted for Poxn have no p-es. Ubiquitous expression of Poxn leads to supernumary p-es and induces the transformation of trichoid sensilla to p-es.

In some of these mutants, new p-es induced by Poxn overexpression are found in positions where no trichoid sensilla are normally found. The new p-es are derived from transformed chordotonal neurons (internal stretch neurons). The induction of new p-es are accompanied by the induction of cut, a gene not normally expressed in chordotonal neurons. Both cut and Poxn are normally expressed in p-es neurons. Although cut is considered to be downstream of achaete-scute, it is apparent that cut is also responsive to Poxn. This illustrates the importance of cross-regulatory interactions in cell fate determination. The pathways are not linear (Vervoort, 1995).

The function of Poxn in the development of the larval peripheral
nervous system (PNS) and in other developmental
aspects has been analyzed using a loss-of-function mutant of Poxn. In addition to the transformation of p-es
into m-es organs in the mutant embryo, the external
structure of the trichome-like sensilla (hairs) misdifferentiates
into that of the campaniform-like sensilla (papillae)
in the second and third larval instars. Poxn is expressed in a cell associated with
the external structure of the trichome-like sensilla in the
first and second instar larvae. These results imply that
Poxn is required in two distinct steps in the development
of the larval PNS: (1) development of the larval p-es organs
during embryogenesis and (2) re-formation of larval
sensory hairs after each larval molt. In addition to
its expression in the developing PNS, Poxn is also expressed
in concentric domains of the leg and antennal
imaginal discs of early third instar larvae, and in the region
of the wing disc that will form the wing hinge. The
loss of Poxn function results in defects of segmentation
of the tarsus and antenna and in a distortion in the wing
hinge. These results indicate that the Poxn gene plays
crucial roles in the morphogenesis of the appendages, in
addition to its role in the early specification of neuronal
identity (Awasaki, 2001).

Various types of sensory organs are arranged in a highly
reproducible pattern on the thoracic and abdominal segments
of Drosophila embryo and larva. In the wild type,
the thoracic (T) segments bear two p-es organs, a lateral
kolbchen (lk) and a ventral kolbchen (vk).
The abdominal (A) segments also bear two p-es organs,
a hair (h3) and a papilla (p6). The external sensory structures were examined in the first instar larva of
the Poxn- null mutant, Poxn70. In the T segments, the lk
is missing and an extra papilla-like structure forms in a
more ventral position, just dorsal to p4. The vk is also
missing and an extra papilla forms more dorsally, adjacent
to p3. In the A segments, the ventral p-es
organ p6 is absent and a new hair forms near the position of h1, while the dorsal p-es organ h3 is substituted
by a papilla-like structure at a position dorsal to h2. These results confirm the observations on
homozygous Df(2R)WMG embryos, where the Poxn locus
is deleted, and demonstrate
that Poxn is involved in the development of the
external structures of larval p-es organs (Awasaki, 2001).

In order to determine whether the transformation of p-es
organs in Poxn mutants affects the number of neurons as
well as the external morphology of the organs,
the development of the embryonic PNS was examined with a neuron specific antibody, MAb22C10. Peripheral neurogenesis is essentially complete and
the arrangement of the neurons is highly invariant in the
mid-embryonic stage. In the meso- and meta-thoracic hemisegments (T2 and T3) of the wild-type embryos, the two p-es organs, vk and lk, are innervated
by three neurons, les3 and v'es3. In the Poxn- mutant, the
les3 neurons are substituted by a single neuron with a
solely extending dendrite. The v'es3 neurons are also
missing and instead a single neuron and an accompanying
md neuron are arranged just below the lateral cluster.
In the first to seventh abdominal hemisegments (A1-A7)
of wild-type embryos, two p-es organs, a hair (h3) and a
papilla (p6), are each innervated by two neurons, des2
and v'es2, respectively. In the Poxn- mutant, the des2
neurons are missing in the dorsal cluster. Instead, a single
neuron forms dorsal to lch5. Likewise the v'es2 neurons
are substituted by a single es neuron accompanied
by a md neuron, at a position near that of the lesA and
ldaA neurons. Thus, in the Poxn- mutant, the neurons
that normally innervate the p-es organs are absent, and
single neurons are found associated to the transformed
external structures (Awasaki, 2001).

It was also noted that the axons of the neurons associated
to the transformed structures fasciculate with those of
the nearest neurons, rather than retracing the pathway
that p-es neurons would normally have followed (Awasaki, 2001).

The SMCs of the p-es organs arise in stage 11 and
they and their progeny were monitored using an enhancer
trap line A37 or anti-Cut antibody. The expression pattern of A37 or
Cut in early steps of PNS development were compared between the
wild-type and Poxn70 /Poxn70 embryos. There are no differences in the pattern between them, indicating that the early steps in PNS development are normal in the mutant (Awasaki, 2001).

This loss of function analysis supports the idea that Poxn
plays a central role in the specification of the p-es organs.
The inactivation of Poxn transforms
the external structures of p-es organs into those of
m-es organs. In the absence of Poxn the number of p-es
neurons is correspondingly reduced from 2-3 to 1, consistent
with the observation that the mutation of Poxn results
in a transformation of poly-innervated gustatory
bristles into mono-innervated tactile bristles in the adult. Thus, the gene Poxn is functionally required for the development of both external
and internal structures of p-es organs in embryos as
well as in adults (Awasaki, 2001).

The position of the transformed organs is not the
same as that of the original ones, indicating that the identity
of the sensory organs influences the positions where
they will eventually differentiate. In wild-type embryos,
the thoracic p-es neurons (les3 and v'es3) are present in
the lateral and ventral clusters, respectively, while the
abdominal p-es neurons (des2 and v'es2) belong to the
dorsal and ventral clusters. In the mutants,
all transformed es neurons are present in the lateral clusters (Awasaki, 2001).

The early steps in PNS development, such as the formation
of the SMCs and their progeny, are normal in the
mutant. Therefore, the misplacement of the transformed
es organs occurs during later development, most probably
as a result of the change in organ identity that
abolishes the programmed migration. The transformed
SMCs will therefore keep their lateral position, and the
organs will correspondingly appear displaced relative to
the expected position of the untransformed p-es organs.
A similar situation has been documented in the case of
the chordotonal (ch) organs. The lateral ch organs (lch5)
in the abdominal segments form dorsolaterally and migrate
to a lateral position during their development. The homologous
thoracic organs, lch3, do not undergo this migration
and remain dorsolateral. When the precursors of
the lch5 are transformed into those of es organs by the
ubiquitous expression of Cut, they keep their original position
and develop in a dorsolateral position. Thus, neural specification genes such as cut and Poxn, in combination with segmental identity genes
such as Ubx, are involved
in the final pattern of the sensory organs: the pattern
depends not only on the position where precursors form,
but also on subsequent migration during morphogenesis (Awasaki, 2001).

The external structure of es organs were examined in the
second and third instar larvae of the Poxn- mutants to
confirm the above results. Surprisingly, it was found that
the external structures of es organs in the second and
third instar mutant larvae are different from those in the
first instar larvae. In addition to the transformation of the
p-es into m-es organs, all hairs disappear and are replaced
by papilla-like structures both in T and A segments.
The positions of the organs, however, do not
change. This indicates that Poxn is required for
the formation of hair-like external structures after the
first instar (Awasaki, 2001).

To confirm the involvement of Poxn in hair formation,
its expression during larval life was examined. Anti-Poxn
immunostaining shows that Poxn is expressed
in a cell closely associated with each hair, presumably
the hair-secreting trichogen cell, during the first and second
instars. This supports the idea that Poxn
plays a role in specifying the external structure of larval
hairs after each molt (Awasaki, 2001).

At each molt, the entire cuticle of the insects, including
many specialized cuticular structures such as external
sensory organs, is shed and has to be rebuilt again. During
the reconstruction process, the trichogen and tormogen
cells of the mechanosensory hairs synthesize material
for the formation of the new shaft and socket, respectively. In
the absence of Poxn function all hair shafts disappear and
are replaced by papilla-like structures in the second and
third instar larvae. Poxn is expressed during the first and second instars in individual cells closely associated with each hair, possibly the trichogen
cells. These observations indicate that Poxn is required
for the reconstruction of the hair shaft after each
molt, although it is unclear whether the papilla-like
structure in the mutant results from a transformation
from hair into papilla or from the loss of the hair structure
itself. First instar mutant larvae do not show this defect
in hair structure, indicating that the formation of hair
shaft during embryogenesis does not need Poxn. Thus,
the initial differentiation of hair shafts during embryogenesis
and their reformation after each larval molt appears
to depend on distinct mechanisms (Awasaki, 2001).

The transformation of the external structures of larval
es organs has been reported in the analysis of the BarH1
and BarH2 genes. When both
Bar genes are deleted, the papillae are transformed into
hairs, while the ubiquitous expression of one of them
suffices to produce the reciprocal transformation of hairs
into papillae. These results have been documented on
embryos, however, and it is not known whether the
BarH genes are also involved in the reconstruction of the
hair/papilla structure after molting. An examination of
possible interactions between Poxn and BarH at various
stages would be interesting (Awasaki, 2001).

In order to discover other functions of Poxn, the pattern of expression of Poxn was examined in imaginal discs. In the leg discs of mid-third instar larvae,
Poxn is expressed in two concentric circles in
a region corresponding to the prospective tarsus. This
expression is transient and disappears in the late third instar
(wandering) larvae. In late third instar larvae, Poxn
expression is also observed in the developing adult chemosensory
bristles in the leg discs (Awasaki, 2001).

In the eye-antennal discs, Poxn is expressed at the
mid-third instar larval stage in the antennal but not in the
eye region. The pattern of expression is very
similar to the pattern in the leg discs: two concentric circles
in the region of the prospective arista, which is homologous to the leg tarsus. This expression is also transient and disappears by the late third larval stage.
In the wing discs of mid-third instar larvae, Poxn is
expressed in four stripes corresponding to regions of the
prospective wing hinge area, in a quadrant pattern. This expression is maintained at least until mid pupal stages. As the dorsal and ventral cells of the
wing disc become faced during early pupation, the dorsal
and ventral regions expressing Poxn become superimposed
and the labeling appears localized to two regions
of the putative wing hinge. In the anterior margin
of the wing disc, Poxn is also expressed in two
rows corresponding to the formation of the adult chemosensory
bristles (Awasaki, 2001).

The pattern of expression of Poxn in leg discs suggests
that Poxn might play a role in leg morphogenesis. The morphology of the mesothoracic leg of Poxn- mutants and of wild-type flies was compared. The tarsus is normally composed of five segments, T1-T5 (proximal to
distal), which are separated from each other by joints. In the mutants, the tarsus is reduced to three segments. The reduction in the number of tarsal segments results
from a fusion rather than from a loss of the segments.
The intermediate segment of the mutant tarsus includes
the bristle patterns of the T2 and T3 segments of wild-type
flies, which is marked by a gradual increase of bristle
length from proximal to distal in each tarsal segment. Also, in many cases, an incomplete ball and socket is seen where the prospective joint should
have formed. However, the intersegmental membrane is never seen on the ventral
side. Thus, T2 and T3 segements would be replaced by a
single T2/3 segment in the mutants (Awasaki, 2001).

A similar situation occurs in the distal segment of the
mutant tarsi, where the pattern of bristles corresponds to
the juxtaposition of the patterns in the normal T4 and
T5 segments. It is concluded that T4 and T5 are fused into a single T4/5 segment. In
this case, however, no incomplete joints are seen. Thus
Poxn is required for formation of the joints between T2
and T3, and between T4 and T5 (Awasaki, 2001).

The length of the tarsal segments is reduced in the
mutants. The T2/3 segment is reduced by 34% relative to the combined length of T2+T3, while for T4/5 the reduction is about 56% relative to that of T4+T5. A
20% reduction is also observed in the mutant T1 segment.
The size or patterning around the circumference of
the tarsus appears normal, based on cuticular and bristle
pattern. Other leg segments (tibia, femur, trochanter and
coxa) are unaffected in the mutants (Awasaki, 2001).

The effect of the Poxn- mutation on the
morphology of the antenna was examined. In wild-type flies, the antenna
comprises three segments plus the arista, which
stands on a basal cylinder. The basal cylinder,
which consists of two small segments, has been shown
to be homologous to segments T2-T4 of the tarsus. In the mutant,
the joint between the basal cylinder and arista does not
form properly. The other structures of the antenna
are not affected in the mutants. Thus, the loss of
Poxn function causes homologous defects in the leg and
antenna (Awasaki, 2001).

The wing structure at the hinge region in
wild-type and mutant flies was compared. Two regions,
which correspond to the regions of expression of Poxn
in the prospective wing, are deformed in the mutants.
Anteriorly, the proximal part of the radius is thick and
shortened in the mutants. Posteriorly, the first vannal vein and the postcubitus are fused and the region anterior to the alula is reduced and misformed (Awasaki, 2001).

Leg formation occurs through concentric folding of leg
discs and subsequent segmentation of epithelia of leg
discs along the proximodistal axis to generate concentric
domains. Leg segmentation requires repeated subdivision occurring in multiple phases. The decapentaplegic and wingless genes encoding secreted
signaling molecules are expressed dorsally and
ventrally, respectively, along the anteroposterior compartment
boundary. They define the concentric expression
of Distal-less and dachshund in the leg disc. Dll is expressed in the
distal region of leg discs and is required for the development
of all distal structure other than coxa. Several
genes, such as aristaless, spineless, bric a brac and
BarH1 and BarH2, act downstream
of Dll to establish the tarsal segments. The evidence that
Poxn is expressed in the leg in regions that correspond
exactly to the tarsal region where the morphological defects
were seen in the mutant indicates the involvement
of Poxn in tarsal formation. Poxn expression is abolished in the Dll mutants, supporting the view
that Poxn also acts downstream of Dll in tarsal formation (Awasaki, 2001 and references therein).

Similar losses of tarsal joints or segment borders
are known in other mutants such as four-jointed and bab. The fj minus mutation
causes a fusion of T3 to T2 in the tarsal segment,
whereas one class of bric a brac minus mutations causes a
complete fusion of T4 to T5 and a partial fusion of T4 to
T3 and of T3 to T2. These phenotypic similarities present
an interesting problem as to whether these genes act
upstream or downstream of Poxn. The epistatic relationships among these genes is currently being examined (Awasaki, 2001).

The wing of Drosophila is composed of two regions, the
proximal hinge and the distal wing blade.
The proximodistal patterning of the wing differs from
that of legs. Different spatial and temporal interactions between
Notch, wg and vestigial specify proximal and distal
pattern elements of the wing. After specification of a
wing primordium, cells that are exposed to the activity
of both wg and vg will become wing blade and those that
are continuously under the influence of wg alone will develop
as hinge. Another gene, nubbin, is also required
for the proximodistal specification in the wing
disc. The expression of nub is restricted to the wing pouch
(the region of the wing disc that corresponds to the prospective
wing blade) and mutations in the gene cause a
severe reduction of the wing with transformation of distal
elements into proximal ones. Poxn is required for the formation of the wing hinge region. Preliminary experiments have shown that the region
of expression of Poxn is expanded in nub mutants, suggesting that the expression of the nub gene in the distal region is necessary to
restrict the expression of Poxn to the proximal region (Awasaki, 2001).

The Pax gene family consists of tissue-specific transcriptional
regulators that contain the DNA-binding 'paired'
domain. Numerous Pax genes have been identified in
various animals. They are classified into four groups
each of which shares a specific motif and a highly conserved
paired domain. The Poxn gene
belongs to group II. In addition to Poxn, the other known member of Pax group II in Drosophila is shaven, which plays a role in the development
of the PNS. Interestingly, shaven is also known
to function in the differentiation of the shaft. The overall structure of shaven is closely related to that of the other group II genes, whereas Poxn is more distantly related to the other
genes of this group. It is therefore tempting to speculate that the functions apparently shared between Poxn and shaven, i.e. their role in
hair formation, may correspond to conserved ancestral
functions, while the function uniquely associated to
Poxn, namely the specification of p-es organs, may have
been acquired more recently by this gene. In this context,
it will be interesting to know how Poxn acquired its specific
functions in the development of the wing hinge and
in the segmentation of tarsus and antenna. The identification
and analysis of genes related to Poxn and shaven
in other insects may give answers to this question and
shed some light on the relationship between gene variation
and the evolution of development (Awasaki, 2001).

The Pox neuro (Poxn) gene of Drosophila plays a crucial role in the development of poly-innervated external sensory (p-es) organs. However, how Poxn exerts this role has remained elusive. This paper analyzes the cell lineages of all larval p-es organs, namely of the kolbchen, papilla 6, and hair 3. Surprisingly, these lineages are distinct from any previously reported cell lineages of sensory organs. Unlike the well-established lineage of mono-innervated external sensory (m-es) organs and a previously proposed model of the p-es lineage, this study demonstrate that all wild-type p-es lineages exhibit the following features: the secondary precursor, pIIa, gives rise to all the three support cells - socket, shaft, and sheath, whereas the other secondary precursor, pIIb, is neuronal and gives rise to all neurons. It was further shown that in one of the p-es lineages, that of papilla 6, one cell undergoes apoptosis. By contrast in Poxn null mutants, all p-es lineages have a reduced number of cells and their pattern of cell divisions is changed to that of an m-es organ, with the exception of a lineage in a minority of mutant kolbchen that retains a second bipolar neuron. Indeed, the role of Poxn in p-es lineages is consistent with the specification of the developmental potential of secondary precursors and the regulation of cell division but not apoptosis (Jiang, 2014).

GENE STRUCTURE

Genomic length - 8 kb

Bases in 5' UTR - 747+

Exons - four

Bases in 3' UTR - 338

PROTEIN STRUCTURE

Amino Acids - 425

Structural Domains

Poxn and Pox-m share presence of paired domains with paired but unlike paired they have no homeodomains (Bopp, 1989).

Evolutionary Homologies

Poxn and POX-M, paired box regions, are homologous to the paired box regions of paired and of the two gooseberry genes. Unlike the gooseberry genes and the engrailed-invected pair, Poxn and pox-m are not linked and therefore have differing developmental roles. pox-m is involved in mesodermal differentiation.

The many paired-box containing genes in mice fall into six classes. The Drosophila Poxn by itself falls into class V, while POX-M falls into class I with human Pax-1 and HuP48. Drosophila Paired, Gooseberry-proximal and Gooseberry-distal fall into class II (Walther, 1991).

Pax proteins play a diverse role in early animal
development and contain the characteristic paired
domain, consisting of two conserved helix-turn-helix
motifs. In many Pax proteins the paired domain is fused
to a second DNA binding domain of the paired-like
homeobox family. By amino acid sequence alignments,
secondary structure prediction, 3D-structure comparison,
and phylogenetic reconstruction, the relationship
between Pax proteins and members of the Tc1
family of transposases, which possibly share a common
ancestor with Pax proteins, has been examined. It is suggested that the DNA
binding domain of an ancestral transposase (proto-Pax
transposase) was fused to a homeodomain shortly after
the emergence of metazoans about one billion years ago.
Using the transposase sequences as an outgroup the early evolution of the Pax proteins was examined. This
novel evolutionary scenario features a single homeobox
capturing event and an early duplication of Pax genes
before the divergence of porifera, indicating a more
diverse role of Pax proteins in primitive animals than
previously expected (Breitling, 2000).

An attemp has been made to reconstruct the phylogeny
and to reliably root the phylogenetic tree of Pax proteins. Since homeodomains,
which have been compared for that purpose, are only present in some of the
Pax proteins and are conspicuously absent in the
PaxA/neuro and Pax1-9 group, the analysis was restricted
to the paired box itself. This was facilitated by the introduction of a novel outgroup. Comparison of the X-ray structures of the paired box of Drosophila Paired
(1PDN) and human Pax6 (6PAX) within the database of
3D-structures has revealed that the N-terminal subdomain (PAI domain) is closely related to the DNA binding domain of Tc3 transposase of
Caenorhabditis elegans (1TC3). A general similarity between
transposase DNA binding domains and the paired
domain has been reported and their
structural relationship has been observed during the
analysis of the transposase structure.
Initial Blast searches identified a group of transposases
from C. elegans whose DNA binding domain seems to
be more closely related to the paired box than to most
other transposases. The DNA binding domain of these
C. elegans transposases (proteins K03H6.3, W04G5.1,
F26H9.3, F49C5.8, and C27H2.1; accession numbers
T33011, T26169, T21438, T22423, and T19530) shows
highly significant similarity only to Bmmar1, a transposase
from Bombyx mori [accession number AAB47739, E-score
(E)=2e-27 compared to K03H6.3], and to many Pax
proteins (e.g. Hydra magnapapillata Pax2/5/8, E=9e-05;
Phallusia mammilata Pax6 E=3e-04; or Paracentrotus
lividus Pax1/9 E=6e-04). The DNA binding domains of
other transposases yield E-scores worse than 1e-03 (e.g.
Anopheles albimanus transposase AAB02109, E=9e-03).
It is supposed that the transposases of C. elegans and
B. mori might represent molecular fossils (proto-Pax)
from the time before a homeobox capturing event took
place, during which the catalytic domain of the transposase
was lost and the DNA-binding domain was fused to a
homeobox yielding the first PAX protein. If this is
indeed the case, the proto-Pax transposases should also
contain the C-terminal subdomain (RED domain) of the
paired box. This subdomain is less conserved among Pax
proteins than the PAI domain and does not show significant homology in sequence alignments between transposases
and Pax proteins. A secondary structure analysis of the proto-Pax transposases
was performed using a consensus method (Jpred2), which predicted that
they indeed contain two helix-turn-helix motifs, homologous
to both the PAI and the RED domain of Pax proteins (Breitling, 2000).

The observation that the DNA binding domain of
transposases is in fact closely related to the paired box
indicates that it should be possible to use them as an
outgroup in the phylogenetic analysis of Pax proteins to
determine the most likely evolutionary sequence. The transposase sequence (C. elegans K03H6.3, E = 2e-27) with the highest Blast score was
compared to Pax proteins to generate a multiple
sequence alignment of Pax-like transposases using the
JPred2 server. The JPred2 algorithm was also used to
generate a multiple sequence alignment for Pax proteins.
Both alignments were combined and realigned by using
ClustalW. The resulting data set contains
transposases of the Tc1 and mariner families, as well as a
wide range of Pax proteins from all known subgroups. The complete alignment was then used for phylogenetic analysis (Breitling, 2000).

Neighbor-joining and parsimony analysis reliably
subdivides the Pax proteins into five large groups,
which correspond to the classical subfamilies Pax1-9/Pax
meso, PaxD/3-7/Gooseberry/Paired, PaxB/2-5-8/Sparkling,
Pax4-6/Eyeless and PaxA/Pax neuro. The internal
topology of the subfamilies agrees fairly well with the
accepted evolutionary relationship of the organisms. One
exception is the Pax4-6/Eyeless subfamily which is
extremely conserved, so that an unambiguous determination
of the internal branching order was not possible. The
position of Drosophila Eyegone is also unreliable,
because this protein contains only a partial paired domain. In both trees PaxC is significantly
associated with the PaxA/Pax neuro subfamily, although PaxC
carries a homeobox, and PaxA/Pax neuro proteins do not.
Neighbor-joining and parsimony tree reconstruction
place the Pax family within the Tc1 family of transposases,
while it was not possible to identify a single closest relative
of the paired box. The supposed proto-Pax transposases
from C. elegans and B. mori, as identified by Blast searches,
are not reliably placed as a sister-group of the Pax
proteins. This might be due to the general difficulty of
reconstructing well-resolved phylogenetic trees of the
transposase family (Breitling, 2000).

This focus on the paired box as a descendant of a Tc1-
like transposase DNA binding domain allowed for a
reevaluation of the early evolution of the paired domain. These
results show that the evolutionary scenario proposed by
Galliot and Miller (2000) is unlikely to correctly represent
the evolution of Pax proteins. This hypothesis
was based mainly on the assumption that PaxA, which
consists only of a paired box, resembles the probable
ancestor of Pax proteins. Contrary to that idea, the
scenario developed here is based on the assumption that the
paired box is originally derived from a transposase and
indicates that PaxA is probably derived by a secondary
loss of the homeobox of a PaxC-like protein. These observations
also make unlikely the hypothesis that there was
more than one homeodomain capturing event. Furthermore,
they suggest that the first duplication of Pax
proteins occurred before the divergence of the porifera.
This consequently implies that sponges, which lack
nerve cells and most of the organs patterned by Pax
genes in higher animals, already contained (at least) two
Pax genes. The function of these early Pax proteins
remains a mystery (Breitling, 2000).

target of Poxn is a potential target of Paired box neuro. Poxn is expressed in two clusters of cells in each segment, one dorsal and one ventral. The dorsal-most cluster is displaced laterally in the second and third thoracic segments, a pattern typical of the chemosensory organs. The expression of tap follows the same pattern, with two important differences. (1) While Poxn is expressed in the sensory mother cell (SMC) and throughout the lineage until shortly before the progeny undergo differentiation, tap is expressed only at or near the onset of differentiation. Thus tap expression is very transient, lasting probably for less than an hour. (2) While Poxn is expressed in most or all of the progeny of the SMC, tap is expressed in only one cell of each organ (Gautier, 1997).

It has been confirmed that tap depends (directly or indirectly) on Poxn by inducing the ectopic expression of Poxn early during embryogenesis. the overexpression of Poxn has been shown to result in the development of ectopic chemosensory organs, both in the larva and in the adult. Additional cells expressing tap were observed embryos were the ectopic expression of Poxn was induced at 4-6 h after egg laying. Conversely, in embryos homozygous for a deficiency removing Poxn, the expression of tap is completely abolished and largely but not completely so in the CNS. Poxn binds to polytene chromosomes at 74B, the same location that codes for tap (Gautier, 1997).

Targets of Activity

The gene cut is expressed in the external sense organs. This expression differentiates external sense organs from chordotonal neurons. Among the external sense organs, Poxn is expressed in only poly-innervated organs where it inducescut and differentiates these from the mono-innervated organs. Poxn expression does not depend on cut, while pox-m can induce cut in poly-innervated external sex organs. The identity of the cut regulation region controlled by Poxn has been established (Verwoort, 1995).

abd-A regulates the segmental identity of neural elements in the peripheral nervous system. Anti-Poxn stains cells in the PNS that give rise to poly-innervated sensory organs. Some of these stain-accepting cells produce structures that are homologous to one another yet still different from one another, depending on their location (thorax or abdomen). A dorsal row of Poxn-positive cells become kölbchen in the thorax (dorsal pits), but become small sensory hairs in the abdomen. These sense organs differ in both their position and in their differentiation. In thoracic segments T2 and T3, the dorsal Poxn-positive cells migrate to a more ventral position than do those in the abdomen. Both differential migration and the terminal differentiation of these precursors are determined by abdominal-A (Castelli-Gair, 1994).

DEVELOPMENTAL BIOLOGY

Embryonic

Poxn expression is restricted to two neuronal stem cells (neuroblasts) of the CNS, and two sensory mother cells in the peripheral nervous system (Dambly-Chaudière, 1992). The pattern of pox- neuro expression becomes more complicated as more neurons are generated. It appears however that cells expressing Poxn are clonally related (Bopp, 1989).

The expression in the PNS is confined to precursors of poly-innervated external sensory organs, the chemically sensitive campaniform sensilla. There is additional expression in gnathal segments (Dambly-Chaudière, 1992).

Larval

Expression of Poxn in the wing disc is restricted to the sensory mother cells of the poly-innervated sense organs, suggesting that Poxn also determines the lineage of poly-innervated adult sense organs (Dambly-Chaudière, 1992).

The gene Poxn codes for a transcriptional regulator that specifies poly-innervated (chemosensory),
as opposed to mono-innervated (mechanosensory), organs in Drosophila. The ectopic expression
of Poxn during metamorphosis results in a transformation of the morphology and central projection
of adult mechanosensory organs toward those of chemosensory organs. Poxn also controls the number of neurons. To determine whether Poxn
can transform not only the sense organ precursor cells but also their daughter cells,
the effects of the ectopic expression of Poxn were examined at different stages of the lineage. Poxn can act at a late stage to affect the fate of the undifferentiated neuron (Nottebohm, 1994).

Adult

Overexpression of Poxn induces the morphological transformation of bristles on the adult leg from mechanosensory to chemosensory. The neurons innervating the transformed bristles follow pathways and establish connections appropriate for chemosensory bristles (Nottebohm, 1992). Behavioural tests show that these neurons establish connections appropriate to taste-mediating bristles (Nottebohm, 1994).

The embryonic brain of Drosophila can be subdivided into the
protocerebrum (PC or b1), deutocerebrum (DC or b2) and tritocerebrum (TC or
b3) of the supra-esophageal ganglion and the mandibular (S1), maxillary (S2)
and labial (S3) neuromeres of the sub-oesophageal ganglion. Expression of
engrailed (en) delimits these subdivisions by marking their
most posterior neurons. Because of
morphogenetic processes, such as the beginning of head involution, the
neuraxis of the embryonic brain curves dorsoposteriorly within the embryo.
Accordingly, anteroposterior coordinates will here henceforth refer to the neuraxis
rather than the embryonic body axis (Hirth, 2003).

It was first determined whether Pax2 is expressed in specific domains of the Drosophila brain, by analyzing its expression pattern using in situ hybridization, immunolabelling and lacZ reporter gene expression. Pax2 transcripts initially appear during gastrulation and
at stage 9/10 are observed in a segmentally reiterated pattern of the
developing procephalic and ventral neuroectoderm, with its anteriormost
expression domain located at the future deutocerebral-tritocerebral boundary. Expression of
Pax2 transcripts in the developing brain begins at stage 10/11 and is most prominent in a longitudinal stripe at the medial part of the protocerebrum and in a transversal stripe at the posterior border of the deutocerebrum. Immunolabelling with a Pax2-specific polyclonal antibody reveals that Pax2 protein distribution resembles that of Pax2 transcripts, as
does a Pax2-lacZ reporter gene expressing ß-galactosidase. In addition to its expression in the developing anterior brain, Pax2 expression is also seen in six to eight cells located at the lateral margin of each hemisegment throughout the more posterior CNS regions of the sub-oesophageal ganglion and
ventral nerve cord (Hirth, 2003).

To determine the expression of the second Drosophila Pax2/5/8
ortholog, Poxn expression was characterized using immunolabelling
and lacZ reporter genes. Poxn protein is first detected in the
developing brain at the end of germband extension (stage 10/11) in two stripes of the procephalic neuroectoderm, that subsequently become restricted to the
posterior protocerebrum and the posterior deutocerebrum. Poxn
expression in more posterior regions of the CNS also occurs in segmentally reiterated patterns. A
comparison between Pax2 and Poxn expression domains reveals, that Pax2 and Poxn are never co-expressed in the same cells of the CNS. Moreover, and with one exception, expression of Pax2 and Poxn does not occur at a comparable anteroposterior position along the neuraxis.
The exception is in the posterior deutocerebrum where adjacent Pax2 and Poxn expression domains define a transversal domain immediately
anterior to the tritocerebral brain neuromere. This transversal
domain of adjacent Pax2 and Poxn expression is
distinguishable from segmentally reiterated expression in more posterior
regions by the fact that it is the only position along the neuraxis where
expression of both genes coincides with a neuromere boundary. This
transversal domain of adjacent Pax2/5/8 ortholog expression is referred to as the
deutocerebral-tritocerebral boundary (DTB) region (Hirth, 2003).

It is important to note that the DTB is located anterior to the expression domain of the Drosophila Hox1 ortholog labial
(lab), which is expressed in the posterior tritocerebrum.
Moreover, the DTB is located posterior to the expression domain of the
Drosophila Otx orthologue otd in the protocerebrum and
anterior deutocerebrum. Thus, in Drosophila as in vertebrates, a
Pax2/Poxn (Pax2/5/8) expression domain is located between
the anterior otd/Otx2 and the posterior Hox-expressing regions. This
raises the question of whether the DTB in the embryonic Drosophila
brain might have developmental genetic features similar to those observed for the MHB in vertebrate brain development (Hirth, 2003).

In the embryonic vertebrate brain, Otx2 is expressed anterior to and abutting Gbx2. The future MHB as well as the overlapping domains
of Pax2, Pax5 and Pax8 expression are positioned at this
Otx2-Gbx2 interface. To investigate if comparable expression patterns are found in the embryonic fly brain, the
brain-specific expression of the Drosophila Gbx2 ortholog
unplugged (unpg) was determined in relation to that of otd, using immunolabelling and an unpg-lacZ reporter gene that expresses ß-galactosidase like endogenous unpg. The
otd gene is expressed in the protocerebrum and anterior deutocerebrum of the embryonic brain, as well as in midline cells in more posterior regions of the CNS.
Expression of unpg-lacZ in the embryonic CNS is first
detected at stage 8 in neuroectodermal and mesectodermal cells at the ventral
midline, with an anterior limit of expression at the cephalic furrow.
Subsequently, the unpg expression domains in the CNS widen and have
their most anterior border in the posterior deutocerebrum. Double
immunolabelling of Otd and ß-galactosidase reveal that the posterior
border of the brain-specific otd expression domain coincides with the
anteriormost border of the unpg expression domains along the
anteroposterior neuraxis. There is no overlap of otd and
unpg expression in the brain or in more posterior regions of the CNS (Hirth, 2003).

These findings indicate that the otd-unpg interface is positioned
at the anterior border of the DTB. This was confirmed by additional
immunolabelling studies examining unpg-lacZ, otd, Poxn and
en expression in the protocerebral/deutocerebral region of the
embryonic brain. Thus, double immunolabelling of Otd and En confirms that the posterior border of otd expression extends beyond the protocerebral en-b1 stripe into the anterior deutocerebral domain. Labelling Otd and Poxn confirms that the Poxn expression domain of the DTB is posterior to this deutocerebral otd expression boundary. Labelling En and
ß-galactosidase (indicative of unpg expression),
confirms that the anteriormost unpg expression domain overlaps with the en-b2 stripe.
Finally, labelling ß-galactosidase and Poxn confirms that this
anteriormost unpg expression domain overlaps with the Poxn
expression domain of the DTB. Therefore, in terms of overall gene expression patterns, it is found that a transversal domain of adjacent Pax2/Poxn expression
defines the DTB region of the embryonic Drosophila brain.
Furthermore, this region is located between an anterior otd
expression domain and a posterior Hox expression domain. Moreover, it
is located abutting and posterior to the interface of otd and
unpg expression along the anteroposterior neuraxis (Hirth, 2003).

In mammalian brain development, homozygous Otx2-null mutant
embryos lack the rostral brain, including the MHB-specific Pax2/5/8 expression domain, whereas Gbx2 null mutants misexpress Otx2 and Hoxb1 in the brain. Moreover, Otx2 and Gbx2
negatively regulate each other at the interface of their expression domains. To test if
similar regulatory interactions occur in the embryonic brain of
Drosophila, the expression of the corresponding
orthologs was analyzed in otd and unpg mutant embryos.
In otd-null mutant embryos, the protocerebrum is absent because
protocerebral neuroblasts are not specified. Analysis of unpg, en and Poxn expression
in otd-null mutant embryos reveals that the anteriormost border of unpg expression shifts anteriorly into the anterior deutocerebrum, while Poxn fails to be expressed in the deutocerebrum. In contrast to inactivation of otd, inactivation of unpg does not result in
a loss of cells in the mutant domain of the embryonic brain, as is evident from the expression of an unpg-lacZ reporter construct in
unpg-null mutant embryos. Analysis of otd expression in
unpg-null mutants shows that the posterior limit of brain-specific
otd expression shifts posteriorly into the posterior deutocerebrum,
thus extending into the DTB. This was confirmed by additional immunolabelling studies
examining otd, Poxn and en expression in the
protocerebral/deutocerebral region of the embryonic brain in
unpg-null mutants. Double immunolabelling of Otd and En in
unpg-null mutants confirms that the posterior border of
brain-specific otd expression extends posteriorly to the
deutocerebral en-b2 stripe into the posterior deutocerebrum. In addition, double
immunolabelling of Otd and Poxn in unpg-null mutants confirms that the posterior border of brain-specific otd expression extends posteriorly into the Poxn expression domain of the DTB. Moreover, analysis of lab expression in unpg-null mutants shows that brain-specific lab expression shifts anteriorly into the anterior tritocerebrum. Thus, in both Drosophila and mammals, mutational inactivation of otd/Otx2 and unpg/Gbx2 results in the loss or misplacement of the brain-specific expression domains of orthologous Pax and Hox genes. Moreover, otd and unpg appear to negatively regulate each other at the interface of their expression domains (Hirth, 2003).

In vertebrate brain development, the Pax2 gene, and subsequently
the Pax5 and Pax8 genes, are among the first genes expressed
at the Otx2/Gbx2 interface, followed by the overlapping expression of
En1 and Fgf8 genes. Inactivation of Pax2, Pax5, En1 or
Fgf8 results in the loss of the midbrain and cerebellum because of a
failure to maintain development of this brain region.
In Drosophila, no obvious brain phenotypes were seen after mutational
inactivation of Pax2, Poxn, en/inv or the Drosophila Fgf
homolog branchless (bnl). The absence of
brain phenotypes in these mutants contrasts with those observed in the
vertebrate brain following mutational inactivation of the orthologous
Pax2, Pax5, En1 and Fgf8 genes (Hirth, 2003).

Comparative developmental studies in urochordates and vertebrates have led
to the notion that the basic ground plan for the chordate brain consists of a
forebrain/midbrain region characterized by Otx gene expression, a hindbrain
region characterized by Hox gene expression, and an intervening boundary
region characterized by expression of Pax2/5/8 genes. This suggests that a
corresponding, evolutionarily conserved, tripartite organization also
characterized the brain of the last common ancestor of insects and
chordates. A comparison of the brain-specific topology of gene expression patterns
that define this tripartite organization in Drosophila and in mouse
suggests that the vertebrate midbrain/hindbrain boundary (MHB) region
corresponds to the insect deutocerebral-tritocerebral boundary (DTB) region.
If this is the case, one might expect that other patterning genes that
characterize the MHB region are also expressed at the insect DTB. Although
this expectation is fulfilled for the segment-polarity genes en and wingless (wg) in Drosophila, these two genes are
expressed at the borders of all CNS neuromeres, as well as at parasegmental boundaries in the epidermis; hence, their expression may not be indicative of brain-specific requirements (Hirth, 2003).

In addition to remarkable similarities in orthologous gene expression
between insects and chordates, this study also shows that several functional interactions among key developmental control genes involved in establishing the Pax2/5/8-expressing MHB region of the vertebrate brain are also conserved in insects. Thus, in the embryonic brains of both fly and mouse, the intermediate boundary regions, DTB and MHB, are positioned at the interface of
otd/Otx2 and unpg/Gbx2 expression domains. These boundary
regions are deleted in otd/Otx2-null mutants and mispositioned in
unpg/Gbx2-null mutants. Moreover, otd/Otx2 and
unpg/Gbx2 genes engage in crossregulatory interactions, and appear to act as mutual repressors at the interface of their brain-specific expression domains. However, not all of the functional interactions among genes involved in MHB formation in the mouse appear to be conserved at the
Drosophila DTB. Thus, in the embryonic Drosophila brain, no
patterning defects are observed in null mutants of Pax2, Poxn, en or bnl. It remains to be seen if these genes play a role in the
postembryonic development of the Drosophila brain (Hirth, 2003).

It is conceivable that the similarities of orthologous gene expression patterns and functional interactions in brain development evolved independently in insects and vertebrates. However, a more reasonable explanation is that an evolutionary conserved genetic program underlies brain development in all bilaterians. This would imply that the generation of structural diversity in the embryonic brain is based on positional information that has been invented only once during evolution and is provided by genes such as otd/Otx2, unpg/Gbx2, Pax2/5/8 and Hox, conferring on all bilaterians a common basic principle of brain development. If this is the case, comparable orthologous gene expression and function should also characterize embryonic brain development in other invertebrate lineages such as the lophotrochozoans. This prediction can now be tested in lophotrochozoan model systems such as Platynereis or Dugesia (Hirth, 2003).

Taken together, these results indicate that the tripartite ground plan that characterizes the developing chordate brain is also present in the developing insect brain. This implies that a corresponding tripartite organization already existed in the brain of the last common urbilaterian ancestor of insects and chordates. Therefore, an urbilaterian origin of the tripartite brain is proposed (Hirth, 2003).

Reproduction in higher animals requires the efficient and accurate display of innate mating behaviors. In Drosophila, male courtship consists of a stereotypic sequence of behaviors involving multiple sensory modalities, such as vision, audition, and chemosensation. For example, taste bristles located in the male forelegs and the labial palps are thought to recognize nonvolatile pheromones secreted by the female. A putative pheromone receptor, GR68a, is expressed in chemosensory neurons of about 20 male-specific gustatory bristles in the forelegs. Gr68a expression is dependent on the sex determination gene doublesex, which controls many aspects of sexual differentiation and is necessary for normal courtship behavior. Tetanus toxin-mediated inactivation of Gr68a-expressing neurons or transgene-mediated RNA interference of Gr68a RNA leads to a significant reduction in male courtship performance, suggesting that GR68a protein is an essential component of pheromone-driven courtship behavior in Drosophila (Bray, 2003).

Upon analysis of about a quarter of the 70 Gr genes, a Gr gene, Gr68a, was identified exhibiting the hallmarks of a putative pheromone receptor. In adults, Gr68a is exclusively expressed in neurons of about ten male-specific taste bristles in the forelegs. No expression is observed in females or any other organ or structure of males. Identical β-gal or GFP expression patterns were observed with four independent transgenic p[Gr68a]-Gal4 driver lines, indicating that male-specific expression reflects an intrinsic property of the Gr68a promoter. To verify that the β-gal-positive cells are indeed neurons and not support cells associated with taste bristles, antibody staining was performed; β-gal immunoreactive cells were found to have the typical structure of sensory neurons and express ELAV protein, a pan-neuronal marker not expressed in other cell types. To verify that the Gr68a gene is expressed in one of the chemosensory neurons and not in the single mechanosensory neuron present in taste bristles, its expression was analyzed in a pox-neuro (poxn) mutant background. Poxn is necessary for specification of chemosensory neurons, and poxn mutant flies show a complete transformation of all chemosensory neurons into mechanosensory neurons. Indeed, the p[Gr68a]-Gal4 driver is not expressed in these flies: this confirms that Gr68a is expressed in chemosensory neurons in the male foreleg (Bray, 2003).

Control of bract formation in Drosophila: poxn, kek1, and the EGF-R pathway

In Drosophila, the sensory organs are formed
by cells that derive from a precursor cell through a fixed
lineage. One exception to this rule is the bract cell that
accompanies some of the adult bristles. The bract cell is
derived from the surrounding epidermis and is induced
by the bristle cells. On the adult tibia, bracts are associated
with all mechanosensory bristles, but not with
chemosensory bristles. The differences between chemosensory and mechanosensory lineages are controlled by the selector gene pox-neuro (poxn). This study shows that poxn is also involved in suppressing bract formation near the chemosensory bristles. The gene kek1, described as an inhibitor of the EGF-R signaling pathway, has been identified in a screen for poxn downstream genes. kek1 can suppress bract formation and can interfere with other steps of sensory development, including SMC determination and shaft differentiation (Layalle, 2004).

Misexpression of poxn at a late
stage of mechanosensory bristle development has no
effect on the morphology of the organ, but results in a
suppression of bract formation. Misexpression of poxn
and alteration of the EGF-R pathway affect bract formation
during the same time window. It is concluded that
poxn is responsible for the absence of a bract near the
organs where it is expressed (Layalle, 2004).

kek1, a gene defined as an inhibitor of the EGF-R signaling pathway, is represented in a subtractive library enriched in genes that are specifically
expressed in the chemosensory lineage. kek1 is
not expressed in cells of the mechanosensory lineage at
the time when bract induction takes place, and is expressed
at a high level by the outer cells of the chemosensory organs. Its presence in the subtractive library, and differential pattern of expression between mechanosensory and chemosensory lineages, make kek1 a putative target of poxn (Layalle, 2004).

This point was confirmed by demonstrating that the
expression of kek1 is modified following ectopic expression
of poxn. Specifically, the ubiquitous expression of
poxn results in the activation of kek1 expression in the
outer cells of the mechanosensory lineage, where kek1
is normally silent. The activation of kek1 in mechanosensory
cells is not complete in the experimental conditions that were used.
It should be noted, however, that the repression of
bract formation is also partial, suggesting that the overexpression
of poxn is not complete. Altogether, this
dataset reveals that kek1 is a target of poxn (although
not necessarily a direct one) (Layalle, 2004).

The difference of expression of kek1 in the chemosensory
and mechanosensory lineages, and the role of kek1
in modulating the EGF-R pathway, suggest a role for this
gene in the control of bract formation. kek1 mutants do
not show any abnormality in bract formation or in sensory
organ development, however. More generally, the
complete viability and wildtype phenotype of flies deleted
for kek1 is a surprise, given
the importance of the EGF-R pathway in many aspects of development (Layalle, 2004).

One obvious explanation for this absence of phenotype
in kek1 mutant flies would be the existence of a
functional redundancy between kek1 and another inhibitor
of the EGF-R pathway. This possibility is supported
by the identification of five kekkon-like genes in the Drosophila genome. Therefore this study relied on a gain-of-function analysis to decide whether kek1 might play a role in the control of bract induction (Layalle, 2004).

The EGF-R pathway has been implicated in the formation
of the precursor cells for at least some of the macrochaetae
on the notum. Since kek1 acts as an inhibitor of the EGF-R signaling pathway in ovary development, it might also inhibit this pathway in the notum and thereby interfere with the determination of macrochaetae. The overexpression of kek1 eliminates those macrochaetae that are most dependent on EGF-R signaling (Layalle, 2004).

Macrochaetae suppression was observed when the
expression of kek1 was forced in the proneural cluster
(using the sca-Gal4 driver), but not when its expression
was forced after the SMC had been determined (using
the neu-Gal4 driver). This shows that kek1 interferes
with the formation of the precursor cells but not with
subsequent steps of the lineage. It is concluded that, with
respect to SMC determination, kek1 acts as an inhibitor
of the EGF-R pathway, much as it does in the ovary.
kek1 is expressed in the notum region of third instar wing discs. It may be, therefore, that kek1 plays a role in defining the
position where SMCs are formed, or in defining the time
window during which they are determined. The expression
of kek1 in wing discs is very dynamic, however, and
it has not been possible to determine whether this expression
overlaps that of the proneural genes during normal development (Layalle, 2004).

The overexpression of kek1 induces a loss of mechanosensory
shafts in the legs. At the latest
step of the lineage the socket cell was found to express kek1 at a
high level, whereas the shaft cell does not. The loss of
shafts could therefore be due to a transdetermination of
shaft towards socket fate. No socket
duplication was observed however, and anti-Cut labeling demonstrated
the absence of one of the two support cells at a
frequency similar to that of shaft disappearance. It is concluded that the shaft cell has been lost rather than transformed (Layalle, 2004).

The ectopic expression of kek1
can prevent bract induction near mechanosensory bristles.
This observation is entirely consistent with the idea
that the control of kek1 expression contributes to the
control of bract formation. In the sca-Gal4 line, bracts
may be absent even when a shaft is formed, suggesting a
direct effect of kek1 on bract formation. Since in this
line kek1 expression is driven not only in the mechanosensory
lineage but also in epidermal cells, it may be that this epidermal expression contributes
to bract suppression. Whatever the case, the effect demonstrates that kek1 is capable of interfering with bract formation (Layalle, 2004).

The effect of Kek1 on EGF-R signaling has been
shown to involve a direct interaction between the
extracellular domains of the two proteins. At least part of the effect may be mediated
by heterodimerization, implying that the two genes are expressed in the same cell. The observation that kek1 is expressed in the chemosensory support cells and affects bract formation by ectodermal cells suggests that the Kek1 protein
may also interfere with the functioning of EGF-R
proteins carried by an adjacent cell (Layalle, 2004).

Bract induction involves the activation of the EGF-R
pathway in an epidermal cell, presumably through the
expression of the EGF-R ligand, Spitz, by the outer
cells of the sensory lineage. In the case of
chemosensory lineages, or after ectopic expression of
poxn at a late stage of the mechanosensory lineage,
the presence of Poxn protein activates the expression of kek1 (and presumably of other members of the kek family). The Kek1 protein binds to the EGF-R and
prevents the formation of a bract. The expression
of a dominant-negative form of the receptor
mimics this effect. When the dominant-negative is
expressed both in bristle cells and in epidermal cells
the inhibition of bract formation could be due to the
inactivation of the EFG-R in the cells that receive the
Spitz signal, i.e., in the epidermal cells. An
absence of bracts is also observed when the dominant-negative
form of the EGF-R is overexpressed only in bristles cells. In this case, it is proposed that the supernumerary receptors sequester the ligand and thereby prevent bract induction. Ligand sequestration would also account for the absence of bract cells
when the normal EGF-R is overexpressed in the bristle outer cells (Layalle, 2004).

Taste-independent detection of the caloric content of sugar in Drosophila

Feeding behavior is influenced primarily by two factors: nutritional needs and food palatability. However, the role of food deprivation and metabolic needs in the selection of appropriate food is poorly understood. This study shows that the fruit fly selects calorie-rich foods following prolonged food deprivation in the absence of taste-receptor signaling. Flies mutant for the sugar receptors Gr5a and Gr64a cannot detect the taste of sugar, but still consumed sugar over plain agar after 15 h of starvation. Similarly, pox-neuro mutants that are insensitive to the taste of sugar preferentially consumed sugar over plain agar upon starvation. Moreover, when given a choice between metabolizable sugar (sucrose or D-glucose) and nonmetabolizable (zero-calorie) sugar (sucralose or L-glucose), starved Gr5a; Gr64a double mutants preferred metabolizable sugars. These findings suggest the existence of a taste-independent metabolic sensor that functions in food selection. The preference for calorie-rich food correlates with a decrease in the two main hemolymph sugars, trehalose and glucose, and in glycogen stores, indicating that this sensor is triggered when the internal energy sources are depleted. Thus, the need to replenish depleted energy stores during periods of starvation may be met through the activity of a taste-independent metabolic sensing pathway (Dus, 2011).

This taste-independent sugar-sensing pathway has several distinctive characteristics. First, this pathway is specifically associated with a starved state; taste-blind flies execute food-choice behavior after prolonged food deprivation of between 10 and 15 h of starvation. This time frame coincides with the onset of starvation-induced sleep suppression, indicating that these two behaviors might share a common metabolic trigger. Second, the taste-independent pathway operates on a different timescale from the gustatory pathway. Whereas WT flies made a food choice almost instantly, taste-blind flies chose sugars only after the ingestion of food. Third, this pathway responds to the nutritional content of sugars, but not to their orosensory value. Taste-blind flies chose metabolizable sugars over nonmetabolizable sugars and never consumed nonmetabolizable sugars. Furthermore, the fact that WT flies failed to distinguish a metabolizable sugar from a nonmetabolizable sugar, but shifted their preference to the metabolizable sugar after starvation, indicates that the taste-independent pathway is not an artifact associated with taste-blind flies, but functions in WT flies. Finally, the ability to detect the caloric content of sugars correlated under multiple experimental conditions with drops in hemolymph glycemia (Dus, 2011).

These results demonstrate that starvation directs the selection of nutrient-rich foods in the fly in the absence of the gustatory cues. Thus, as previously suggested in mice, postingestive cues can drive feeding behavior independently of gustatory information. The physiological factors that triggered the taste-independent food choices in mice are, however, unknown. In Drosophila, the internal energy state and carbohydrate metabolism play crucial roles in the metabolic sensing of food according to the results. A possible evolutionary purpose of taste-independent metabolic sensing is to ensure that animals select calorie-rich foods to quickly replenish energy, especially in times of food shortage (Dus, 2011).

How do starved sugar-blind flies preferentially ingest metabolizable sugar over nonmetabolizable sugar? It is plausible that sugar-blind flies are equally attracted to and feed on both sugars, but those on nonmetabolizable sugar resume foraging because of the lack of nutritional value in this sugar. These foraging flies are again equally attracted to both sugars, but those on nonmetabolizable sugar continue to forage until they find the correct food substrate. Food choice in this model is mediated by random selection and 'trapping' of the flies on the metabolizable sugar. Alternatively, sugar-blind flies might readily detect the metabolizable sugar without ingesting a large amount of food because nutrient information is rapidly conveyed to the brain within minutes of ingesting food. In this model, the flies select for metabolizable sugar over nonmetabolizable sugar by a metabolic sensor that operates on a fast timescale to mediate discrimination between the two sugar substrates. Tracking and monitoring the locomotor activity and feeding behavior that generates a preference for metabolizable sugar will address this question (Dus, 2011).

It is intriguing to speculate on the molecular nature of the metabolic sensor. This sensor could be expressed in a subset of neural, digestive, or other tissues. Among the organs and cells that have been proposed for their involvement in feeding regulation in the fly are the fat body, the insulin-producing cells (IPC), and the corpora cardiaca/allata complex. These cells may respond to the metabolic value of sugars in circulation, as seen with the glucose-excited and glucose-inhibited neuropeptide neurons in the arcuate nucleus of the mammalian hypothalamus. A model that explains how changes in circulating glucose levels alter the electrical and secretory properties of the hypothalamic glucose-responsive neurons could also describe how metabolizable sugars trigger the metabolic sensor. In mammals, glucose-sensitive cells detect glucose availability by responding to metabolites of glycolytic enzymes such as hexokinase or the energy-sensing AMP-activated protein kinase (Dus, 2011).

Almost all crucial metabolic functions in mammals are also conserved in Drosophila. During the past decade, researchers using the fruit fly as a model system for studying feeding behaviors and feeding-related disorders, including obesity, have shed much light on the molecular mechanisms of metabolism. By revealing the possibility of a metabolic sensing pathway in Drosophila, this study has introduced the possibility of understanding the molecular mechanism underlying this pathway. Identification of the cellular and genetic nature of this sensor might reveal the identity of the master switch that regulates many hunger-driven behaviors (Dus, 2011).

The molecular basis for water taste in Drosophila

The detection of water and the regulation of water intake are essential for animals to maintain proper osmotic homeostasis. Drosophila and other insects have gustatory sensory neurons that mediate the recognition of external water sources, but little is known about the underlying molecular mechanism for water taste detection. This study identified a member of the Degenerin/Epithelial Sodium Channel family, Pickpocket 28 (Ppk28), as an osmosensitive ion channel that mediates the cellular and behavioral response to water. This study used molecular, cellular, calcium imaging and electrophysiological approaches to show that ppk28 is expressed in water-sensing neurons and loss of ppk28 abolishes water sensitivity. Moreover, ectopic expression of ppk28 confers water sensitivity to bitter-sensing gustatory neurons in the fly and sensitivity to hypo-osmotic solutions when expressed in heterologous cells. These studies link an osmosensitive ion channel to water taste detection and drinking behavior, providing the framework for examining the molecular basis for water detection in other animals (Cameron, 2010).

To uncover novel molecules involved in taste detection, a microarray-based screen was perfected for genes expressed in taste neurons. Proboscis RNA from flies homozygous for a recessive poxn null mutation was compared to RNA from heterozygous controls. poxn mutants have a transformation of labellar gustatory chemosensory bristles into mechanosensory bristles, and therefore lack all taste neurons. Whole genome microarray comparisons revealed that 256 of ~18,500 transcripts were significantly decreased in poxn mutants (>2 fold enrichment in control relative to poxn). These included 18 gustatory receptors (representing a 21-fold enrichment in the gene set) and 8 odorant binding proteins (13-fold enrichment) (Cameron, 2010).

In the mammalian gustatory system, ion channels mediate the detection of sour and salt tastes, suggesting that ion channel genes may also participate in Drosophila taste detection. Therefore the expression pattern of candidate taste-enriched ion channels was examined. The putative promoter of one gene, pickpocket 28 (ppk28), directed robust reporter expression in taste neurons on the proboscis. ppk28 belongs to the Degenerin/Epithelial sodium channel family (Deg/ENaC) and these channels are involved in the detection of diverse stimuli, including mechanosensory stimuli, acids and sodium ions. In the brain, ppk28-Gal4 drives expression of GFP in gustatory sensory axons that project to the primary taste region, the subesophageal ganglion. In situ hybridization experiments confirmed that transgenic expression recapitulates that of the endogenous gene, as 48/52 of ppk28-Gal4 neurons expressed endogenous ppk28 (Cameron, 2010).

Previous studies have identified different taste cell populations in the proboscis, including cells labeled by the gustatory receptor Gr5a that respond to sugars and cells marked by Gr66a that respond to bitter compounds. To determine whether these taste neurons express ppk28-Gal4, co-labeling experiments were performed with reporters for Gr5a and Gr66a. These experiments revealed that ppk28 did not co-label Gr5a cells or Gr66a cells, and is thus unlikely to participate in sweet or bitter taste detection. An enhancer-trap Gal4 line, NP1017-Gal4, labels water-sensing neurons in taste bristles on the proboscis and carbonation-sensing neurons in taste pegs. ppk28 is expressed in taste bristles but not in taste pegs. Interestingly, ppk28 showed partial co-expression with NP1017-Gal4, with the majority of ppk28-positive cells containing NP1017-Gal4 (22/30). This correlation suggested the intriguing possibility that ppk28 participates in water taste detection (Cameron, 2010).

To directly investigate the response specificity of ppk28-expressing neurons, the genetically encoded calcium sensor G-CaMP was expressed in ppk28-Gal4 cells, the proboscis was stimulated with taste substances, and activation of ppk28-Gal4 projections were monitored in the living fly by confocal microscopy. ppk28-Gal4 neurons were tested with a panel of taste solutions, including sugars, bitter compounds, salts, acids and water. ppk28-Gal4 neurons showed robust activity upon water stimulation. In addition, ppk28-positive cells responded to other aqueous solutions even in the presence of a wide range of chemically distinct compounds. This response diminished as a function of concentration. Taste compounds such as NaCl, sucrose and citric acid significantly decreased the response. In addition, compounds unlikely to elicit taste cell activity such as ribose, a sugar that does not activate Gr5a cells, N-methyl-D-glucamine (NMDG), an impermeant organic cation and the non-ionic high molecular weight polymer polyethylene glycol (PEG, 3350 average molecular weight), all blunted the response in a concentration-dependent manner. These data demonstrate that ppk28-expressing neurons respond to hypo-osmotic solutions. This response profile is consistent with previous electrophysiological studies that identified a class of labellar taste neurons activated by water and inhibited by salts, sugars and amino acids (Cameron, 2010).

To determine the function of ppk28 in the water response, a ppk28 null mutant was generated by piggybac transposon mediated gene deletion, removing 1.769kb surrounding the ppk28 gene. The water responses of ppk28 control, mutant and rescue flies were examined by extracellular bristle recordings of l-type labellar taste sensilla. These recordings monitor the responses of the four gustatory neurons in a bristle, including water cells and sugar cells. Control flies showed 12.0±0.9 spikes/sec when stimulated with water. Remarkably, ppk28 mutant cells had a complete loss of the response to water (spikes/sec=0.8±0.1). This response was partially rescued by reintroduction of ppk28 into the mutant background (spikes/sec=6.4±1.0), demonstrating that defects were due to loss of ppk28. Responses to sucrose were not significantly different among the three genotypes, arguing that the loss of ppk28 specifically eliminates the water response. These results were confirmed by G-CaMP imaging experiments that monitor the response of the entire ppk28 population. As expected, ppk28-Gal4 neurons in the mutant did not show fluorescent increases to water and transgenic re-introduction of ppk28 rescued the water response. Taken together, the electrophysiological and imaging data demonstrate that ppk28 is required for the cellular response to water (Cameron, 2010).

The detection of water in the environment and the internal state of the animal may both contribute to drive water consumption. To evaluate the degree to which water taste detection contributes to consumption, the behavioral responses were examined of ppk28 control, mutant and rescue flies to water. Drinking time rather than drinking volume was used to monitor consumption due to difficulty in reliably detecting small volume changes. When presented with a water stimulus, control flies drank on average 10.3±1.1 seconds, mutants drank 3.0±0.3 seconds and rescue flies drank 11.5±1.5 seconds. Additionally, control, mutant and rescue flies ingested sucrose equally, showing that ppk28 mutants do not have general drinking defects. Similar defects in water detection were seen when control, mutant and rescue flies were tested on the proboscis extension reflex to water or when genetically ablating ppk28-Gal4 neurons. Although ppk28 mutants lack water taste cell responses and drink less, they still do consume water, arguing that additional mechanisms must exist to ensure water uptake. These experiments reveal that water taste neurons are necessary for normal water consumption. Moreover, they establish a link between water taste detection in the periphery and the drive to drink water (Cameron, 2010).

Whether ppk28 is directly involved in water detection was examined. If ppk28 is the water sensor, then its expression in non-water sensing cells should bestow responsiveness to water. To test this, the Gal4/UAS system was used to ectopically express ppk28 in Gr66a-expressing, bitter-sensing neurons, and taste-induced responses were monitored by extracellular bristle recordings and G-CaMP imaging experiments. For extracellular bristle recordings, responses were recorded from i-type sensilla which contain bitter-sensing, Gr66a-positive neurons but lack water cells. Expression of ppk28 in Gr66a-Gal4 neurons did not significantly affect the response to denatonium (G-CaMP imaging) or caffeine, endogenous ligands for Gr66a-Gal4 neurons. In response to water, Gr66a-Gal4 neurons showed no significant activity consistent with previous studies. Notably, misexpression of ppk28 in Gr66a-Gal4 neurons conferred sensitivity to water, as seen by extracellular bristle recordings and G-CaMP imaging. Moreover, the response was blunted as solute concentration was increased. Both NMDG and sucrose (substances that do not activate Gr66a-Gal4 neurons) produced dose-sensitive response decreases. The finding that both activation by water and inhibition by other compounds are conferred by ppk28 strongly suggests that ppk28 senses low osmolarity (Cameron, 2010).

To determine if ppk28 requires a taste cell environment to function or confers responsiveness to other cell-types, ppk28 was expressed in HEK293 heterologous cells. A FLAG-tagged ppk28 (inserted after amino acid 222 in the extracellular domain) was expressed in HEK293 cells, confirming that the protein was made and trafficked to the cell surface. For calcium imaging experiments, an untagged version of ppk28 was cotransfected with dsRed. Cells expressing the mammalian trpv4 osmo-sensitive ion channel were used as a positive control and cells transfected with the vector alone as a negative control. Cells were grown in a modified Ringers solution at 303 mmol/kg, loaded with Fluo-4 to visualize calcium changes and challenged with Ringers solution of different osmolalities. Cells transfected with vector alone showed a modest increase at 60% osmotic strength, whereas cells transfected with mammalian trpv4 showed fluorescence increases to all hypo-osmotic solutions, as expected. Importantly, cells transfected with ppk28 significantly responded to decreased osmolality, with dose-sensitive responses elicited by osmolalities of 216 and 174 mmol/kg. These experiments reveal that ppk28 bestows sensitivity to hypo-osmotic solutions in a variety of non-native environments and argue that the channel itself senses low osmolarity. This work provides a foundation for future studies of the biophysical properties of channel activation. Moreover, the ability to express ppk28 in heterologous cells and study its function creates the opportunity to compare its mechanism of gating with other Deg/ENaC family members involved in mechanosensation or sodium sensing (Cameron, 2010).

Overall, these studies examined the molecular basis for water taste detection in Drosophila and identified an ion channel belonging to the Deg/ENaC family, pickpocket 28 (ppk28), as the water gustatory sensor. This work demonstrates that an ion channel responding to low osmolarity mediates cellular and behavioral responses to water. Although the taste of water has received relatively little attention as a classic taste modality, water-responsive taste neurons have been described in many other insects, such as the blowfly and mosquitoes, as well as in mammals, such as cats and rats. The identification of ppk28 as a water taste receptor provides a framework for examining water taste detection in other animals, including humans (Cameron, 2010).

Osmosensation is important not only for the detection of external water sources by peripheral neurons but also for monitoring the plasma osmolality by central neurons. Several studies have identified members of the transient receptor potential family as candidate peripheral and central osmosensors, but the role of members of the Deg/ENaC family in osmosensation has received little attention. The finding that ppk28 is an osmosensitive ion channel raises the possibility that Deg/ENaC ion channels may participate broadly in peripheral and central osmosensation (Cameron, 2010).

Boll, W. and Noll, M. (2002). The Drosophila Pox neuro gene: control of male courtship behavior and fertility as revealed by a complete dissection of all enhancers. Development 129: 5667-5681. PubMed ID: 12421707